Rootstock-regulated gene expression patterns associated ... - CiteSeerX

Jensen et al. BMC Genomics 2012, 13:9 http://www.biomedcentral.com/1471-2164/13/9

RESEARCH ARTICLE

Open Access

Rootstock-regulated gene expression patterns associated with fire blight resistance in apple Philip J Jensen1, Noemi Halbrendt1,2, Gennaro Fazio3, Izabela Makalowska4, Naomi Altman5, Craig Praul6, Siela N Maximova7, Henry K Ngugi1,2, Robert M Crassweller7, James W Travis1,2 and Timothy W McNellis1*

Abstract Background: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. Results: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchardgrown apple trees, consisting of ‘Gala’ scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. ‘Gala’ scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while ‘Gala’ on M.27 and B.9 showed the highest rates of necrosis. ‘Gala’ scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown ‘Gala’ scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. Conclusions: Rootstocks had significant effects on the fire blight susceptibility of ‘Gala’ scions, and rootstockregulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

Background Fire blight, the disease caused by the bacterial pathogen Erwinia amylovora (Burrill) [1], is a devastating, systemic disease that occurs in apples and other Rosaceous plants. Control is limited to pruning of infected branches and the use of antibiotics and copper compounds, both of which are only preventative, and are often strictly regulated. In addition, the emergence of streptomycin-resistant strains * Correspondence: [email protected] 1 Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA Full list of author information is available at the end of the article

of Erwinia amylovora has raised questions about the continued use of this control agent [2]. Once established, infection leads to the development of necrotic regions on the leaves, shoots and petals. The infected regions of the plant eventually become brown or black and look as if swept by fire [3]. Severe fire blight outbreaks can result in the destruction of whole orchards. Current production methods have shifted towards high-density plantings on dwarfing or very-dwarfing rootstocks, resulting in greater yields per acre [4]. However many of the dwarfing rootstocks are highly susceptible to fire blight, resulting in greater disease problems.

© 2012 Jensen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The susceptibility of the different rootstocks and scion cultivars to fire blight varies substantially, and there are recommendations against certain combinations in regions particularly prone to fire blight [5]. It has been observed in the field and in the greenhouse that a given cultivar can have different levels of disease resistance depending on the rootstock to which it is grafted [6,7]. In the case of susceptible scion cultivars, it is recommended that they be grafted to resistant rootstocks to reduce susceptibility to fire blight. In the current study we demonstrate that rootstocks can have a significant effect on the resistance of the scion to fire blight. A number of plant genes and pathways have been implicated as playing roles in the response to E. amylovora infection. Several pathogenesis related (PR) genes have been shown to be up-regulated in apple in response to E. amylovora infection [8,9]. Overexpression of NPR1 in apple results in increased PR gene expression and reduced susceptibility to E. amylovora and a number of other pathogens [10]. Norelli et al. [11] identified transcripts that are differentially expressed between control and E. amylovora-infected shoots using suppression subtractive cDNA hybridization. Recently, further studies identified additional transcripts that are differentially expressed during E. amylovora infection of apple leaves [12] and in apple flowers [13]. E. amylovora has been shown to specifically delay the expression of host genes in the phenylpropanoid pathway during infection [14,15]. This pathway leads to the production of anti-microbial compounds as well as lignin formation [16,17]. In addition, a general increase in free carbohydrate levels has also been associated with increased fire blight susceptibility [15]. Genetic analysis in apple is difficult due to its largely self-incompatible nature, high degree of heterozygosity, and large genome. However, the clonal propagation of apples provides an opportunity for genetic analysis of rootstock-regulated phenotypes, such as disease resistance. In a previous study, we used DNA microarrays to examine steady-state gene expression in the shoot tips of healthy, uninfected ‘Gala’ apple scions grafted to seven different rootstocks [18]. Each of the scion/rootstock combinations had a unique phenotype. In the present study, we undertook to identify constitutively expressed genes in ‘Gala’ apple whose expression levels were associated with a rootstock-induced decrease in fire blight susceptibility. Using fire blight resistance ratings from field-grown trees, we were able to mine the microarray data obtained during the earlier study [18] to identify genes and pathways that might be related to the tree fire blight susceptibility status. Previously, we used a similar approach to identify transcripts whose expression levels correlated with tree stature [18].

Page 2 of 17

Methods Plant Material

Trees for the fire blight tests were purchased from Adams County Nursery Inc. (Aspers, PA) and planted in five replica blocks at The Pennsylvania State University Fruit Research and Extension Center, Biglerville, PA. Trees consisting of ‘Gala’ scions grafted to a range of rootstocks were planted and conventionally managed with pesticides to control weeds, fungal diseases and insects in preparation for inoculation. For the greenhouse-grown tree firstgeneration microarrays, fresh bench grafts were grown as described previously [18]. For fire blight susceptibility tests, ‘Crimson Gala’ (Waliser cultivar) on the same seven rootstocks were planted in 2005 at the Fruit Research and Extension Center in Biglerville, PA. The rootstocks were, from the least to the most vigorous, Malling 27 EMLA (M.27), Budagovsky 9 (B.9), Malling 9 Fleuren 56 (M.9F56), Geneva 30 (G.30), Malling 7 EMLA (M.7), Supporter 4 (S.4), Malling Merton 111 EMLA (MM.111). Throughout the text, plants are described as scion/rootstock combinations. For example, a ‘Gala’ scion on an M.7 rootstock is designated as ‘Gala’/M.7. The trees used for the second-generation microarray experiment were from a segregating population from an ‘Ottawa 3’ × ‘Robusta 5’ cross and were grown in an orchard in Geneva, NY [19]. This population had been previously characterized for resistance to fire blight [19]. Fire Blight Susceptibility

For the fire blight tests conducted in Biglerville, PA, actively growing scion shoot tips of three-year old trees were wounded by using scissors to cut across the midribs of the youngest leaves, and a drop of phosphate buffer (10 mM, pH 7) containing 1 × 10 6 cfu/ml of E. amylovora (strain Ea581a or HKN06P1) was placed on the cut surface. The shoot tip was then covered for 24 hours with a plastic bag containing a wet piece of filter paper to maintain a humid environment and promote infection. Necrotic region measurements were taken over the course of the disease progression. Disease severity was calculated as the length of the blighted section of an inoculated shoot as a percentage of the total shoot length. Four replicate trials were conducted, each replicate consisting of 10 trees of each of the 7 scion/rootstock combinations. At least 5 shoots per tree were inoculated. The fire blight susceptibility of the ‘Ottawa 3’ × ‘Robusta 5’ cross progeny used for the second-generation microarray experiment, to two E. amylovora strains (Ea273, Ea2002a), was published previously [19]. The susceptibility data for a third E. amylovora strain (Ea4001a) is unpublished. RNA isolation and microarray analysis

The methods and results for our first-generation microarray, including RNA isolations from greenhouse-grown


trees and microarray analysis, are described elsewhere [18]. The first generation array contained probes designed to detect 55,230 unique transcripts, representing up to 95% coverage of the apple genome. The sequences for all of the contigs used to develop the probes for the arrays can be found at the Gene Expression Omnibus (GEO) dataset website [20]. For the present study, we developed and used a second-generation apple DNA NimbleGen expression microarray that was designed based on our first-generation NimbleGen array [18] and used it to analyze RNA samples isolated from the progeny of the ‘Ottawa 3’ × ‘Robusta 5’ cross grown in Geneva, NY. The expression levels for each tree were analyzed on a single array only, with no biological replicates for any individual tree. The second-generation array was a 12-plex array containing 135,000 probes per plex, representing 26,017 transcripts, enabling us to query a relatively large number of samples. The probes for this array represent a subset of those included in the first-generation array. The second-generation array represents the transcripts with the best-performing probe sets from the first-generation array and includes the transcripts that showed differential expression between any two scion/rootstock combinations. Transcripts that had high variability among their probes were left off of the second-generation array. The five bestperforming probes of the original six probes per transcript were used in the second-generation array to increase the number of different transcripts that could be queried by the second-generation array. DNA microarray analysis on our second-generation microarrays was performed by the Penn State Genomics Core Facility at University Park, PA. Briefly, one microgram of total RNA from each sample was amplified using the Ambion (Life Technologies) Amino Allyl MessageAmp II aRNA Amplification Kit (AM1753) following the manufacturer’s protocol for one cycle amplification. Fifteen micrograms of aRNA was dye coupled with either Cy3 or Cy5 (GE Health Care #RPN5661), as appropriate. Following quenching and cleanup of dye coupling reactions, 1.5 μg of a Cy3 labeled sample is combined with 1.5 μg of a Cy5 labeled sample and fragmented using RNA Fragmentation Reagents (Ambion AM8740) according the manufacturer’s instructions. After fragmentation, samples are dried down completely in a speed-vac and then resuspended in tracking controls and hybridization solution according to the microarray manufacturer’s instructions (Roche NimbleGen). Pairs of samples were hybridized overnight at 42°C with active mixing in a MAUI Hybridization System. Following hybridization, microarrays were washed and scanned according to the manufacturer’s protocol (Roche NimbleGen). Images were burst, gridded, and pair files generated using NimbleScan software. The gene expression data from the hybridization experiments

Page 3 of 17

using the second-generation DNA microarray were normalized using R software and un-adjusted p-values were calculated by regression analysis using R software [21,22]. Multiple regression analysis

Stepwise multiple regression analysis was used to identify genes whose expression levels were related to fire blight severity. The response variable was mean fire blight severity, calculated as the length of blighted section of an inoculated shoot as a percentage of the entire shoot and averaged across 5 replicates for each of 48 (Geneva trees). The explanatory variables were gene expression levels, expressed as arbitrary units of fluorescence intensity, for a set of 60 candidate genes selected based on potential for involvement in fire blight susceptibility. A full stepwise regression model was implemented so that each of the explanatory variables was evaluated in the regression with significance level for staying in the model set at P = 0.15. For both up-regulated and down-regulated candidate genes, separate stepwise regression analyses were computed for fire blight severity data for each of three E. amylovora strains. Stepwise multiple regression analysis were implemented using the REG procedure of SAS 9.2 (SAS Institute Inc., Cary, NC), and the resulting models were evaluated for goodness of fit based on standard regression analysis procedures [23].

Results Rootstock-dependent differences in fire blight susceptibility of ‘Gala’ scions

Significant differences in the relative size of the necrotic regions were observed within 15 days of inoculation of the ‘Gala’ shoot tips with two different strains of E. amylovora (Figure 1). For both strains, ‘Gala’/G.30 and ‘Gala’/M.111 were the least susceptible and ‘Gala’/B.9 and ‘Gala’/M.27 were the most susceptible. Interestingly, strain-dependent differences in fire blight susceptibility were observed for ‘Gala’/M.7 and ‘Gala’/M.9F56 trees. ‘Gala’/M.7 susceptibility to E. amylovora strain Ea581a was similar to that of the most susceptible trees (’Gala’/B.9 and ‘Gala’/M.27), while ‘Gala’/M.9F56 susceptibility to strain Ea581a was similar to that of the most resistant trees (’Gala’/G.30 and ‘Gala’/ MM.111). The results were reversed with E. amylovora strain HKN06P1, with ‘Gala’/M.7 susceptibility being similar to that of the most resistant trees and ‘Gala’/M.9F56 susceptibility being similar to that of the most susceptible trees. E. amylovora strain Ea581a is a moderately virulent isolate, while HKN06P1 is a hypervirulent isolate [24]. Clustering of trees according to phenylpropanoid pathway gene expression

After obtaining fire blight susceptibility ratings for all the ‘Gala’/rootstock combinations used in the study, it became possible to ask whether expression levels of


Ea581a

20

Disease Severity

7 DAI

M.7

15 DAI

c

c

G.30

bc

15

10

Page 4 of 17

a

ab

a

M.111

ab

S.4 B.9

5

M.9F56 0 G.30 100 90 Disease Severity

80 70

MM.111

M.7

Sup.4

M.9F56

B.9

HKN06P1 7 DAI

15 DAI

ab

b

bc

M.27

M.27 7

c

c

c

ab

60

6

5

4

3

2

1

Euclidean distance (Log2)

Highly susceptible

Strain-dependent susceptibility

Least susceptible

Intermediate susceptibility

Figure 2 Clustering analysis of transcripts involved in the phenylpropanoid biosynthetic pathway using complete linkage with Euclidean distance (Log2).

50 40 30 20 10 0 G.30

MM.111

M.7

Sup.4

M.9F56

B.9

M.27

Rootstock

Figure 1 Susceptibility of grafted ‘Gala’ scions on seven different rootstocks to two different strains of E. amylovora. Bars sharing the same letter are not significantly different (Fisher’s LSD).

genes involved in specific biochemical pathways related to fire blight resistance might contribute to the rootstock-regulated fire blight resistance phenotype. Gene expression levels in scions of all the ‘Gala’/rootstock combinations used in this study were previously profiled on a large scale using our first-generation DNA microarray [18], and these microarray data were mined in the present study for genes related to fire blight susceptibility. Because the phenylpropanoid biosynthesis pathway has been implicated in fire blight resistance [11,14], a search of the apple genome was conducted to find the predicted phenylpropanoid biosynthetic pathway genes. A total of 67 transcripts on our first-generation array were identified as belonging to the phenylpropanoid biosynthetic pathway, among other pathways (Additional File 1, Table S1). A complete linkage cluster analysis using the expression values for these genes was conducted to evaluate the potential contribution of the expression levels of genes in this pathway to resistance (Figure 2). There were two main clusters of trees based on the expression patterns of putative phenylpropanoid biosynthetic genes. The two least susceptible scion/rootstock combinations (‘Gala’/

G.30 and ‘Gala’/M.111) were paired in one cluster, along with a branch containing one scion/rootstock combination displaying strain-dependent susceptibility (‘Gala’/ M.7). The second main cluster contained both of the highly susceptible scion/rootstock combinations (‘Gala’/ M.27 and ‘Gala’/B.9) as well as a scion/rootstock combination with strain-dependent susceptibility (‘Gala’/ M.9F56) and the moderately susceptible scion/rootstock combination (‘Gala’/S.4). Thus, the clustering of trees according to phenylpropanoid pathway gene expression closely followed the pattern of susceptibility to the highly virulent E. amylovora strain HKN06P1 (Figure 1). Clustering of trees according to sugar metabolic pathway gene expression

The AraCyc metabolic pathways tool [25] was used to identify 93 Arabidopsis genes involved in sugar metabolism, and the potential homologs of these Arabidopsis genes in the apple genome were identified. A BLAST search of the set of the Arabidopsis genes involved in sugar metabolism to the apple genome resulted in the identification of 227 unique apple coding sequences. A total of 219 of these transcripts were represented on our first-generation array. A complete linkage cluster analysis using the expression values for the 219 identified sugar metabolism genes did not closely follow the pattern for the levels of fire-blight susceptibility for the various scion/rootstock combinations (compare Figures 1 & 3). Instead, the clustering by sugar metabolism gene expression closely resembled clustering based on data for all the transcripts represented on the first-generation array [18].


Page 5 of 17

Fire Blight Susceptibility

M.111 G.30 S.4

Low

High

M.111

M.27

G.30

B.9

M.27 M.7 M.9F56 B.9 10

9

8

7

6

5

4

3

Euclidean distance (Log2)

Highly susceptible

Strain-dependent susceptibility

Least susceptible

Intermediate susceptibility

Figure 3 Clustering analysis of transcripts involved in sugar metabolism using complete linkage with Euclidean distance (Log2).

Identification and categorization of candidate rootstockregulated, fire blight susceptibility-associated transcripts

Constitutive gene expression levels in scions of all the ‘Gala’/rootstock combinations used in this study were profiled on a large scale previously using our first-generation apple DNA microarray [18]. Using the fire blight susceptibility ratings from the field trials, the microarray data were sorted to identify those transcripts whose expression levels correlated with the differences in fire blight susceptibility among the apple trees being studied. A diagram showing the comparisons used to sort the data is shown in Figure 4. Transcripts of interest were selected based on a fold-expression difference cutoff and a statistical strength cutoff. For a transcript to be selected, every possible pairwise comparison between the two least susceptible (‘Gala’/G.30 and ‘Gala’/M.111) and the two most susceptible scion/rootstock combinations (‘Gala’/B.9 and ‘Gala’/M/27) had to have at least a 1.5 fold difference in expression and a q-value of less than 0.05. This selection program resulted in a list of 665 transcripts with higher expression levels in the less susceptible trees (Additional File 1, Table S2), and 25 transcripts with higher expression in the more susceptible trees (Additional File 1, Table S3) for a total of 690 candidate rootstock-regulated, fire blight susceptibility-associated transcripts. The Malus genome BLAST hits and corresponding e-values are included in Additional File 1, Tables S2 and S3. An analysis of the predicted functional categories of all of the candidate rootstock-regulated, fire blight susceptibility-associated transcripts was conducted based on the Arabidopsis thaliana BLASTX hits to the Malus

Each pairwise combination must have an M-value of greater than 0.58 or less than -0.58 The q-value must be < 0.05 for each comparison 665 transcripts UP in trees with Low Susceptibility 25 transcripts UP in trees with High Susceptibility Figure 4 Selection scheme used to identify candidate rootstock-regulated, fire blight susceptibility-associated transcripts based on expression data.

x domestica sequence (BLASTX cutoff 1E-3). The distribution in the functional categories differed from that expected based on the whole transcriptome. These Gene Ontology (GO) enrichment patterns are shown in Table 1. Transcripts of genes predicted to be involved in responses to stress and biotic and abiotic stimuli were disproportionately represented relative to the known apple transcriptome as a whole. A functional annotation of the transcripts in Additional File 1, Table S2 was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) [26] to look for pathways that might be overrepresented among the candidate rootstock-regulated, fire blight susceptibility-associated transcripts. The sequence of every transcript on the array was subjected to KEGG analysis to generate an overall picture of the relative abundance of genes in the KEGG pathways. Similarly, all of the transcripts in Additional File 1, Tables S2 and S3 were analyzed for the KEGG pathways. A chi-square analysis revealed that, for the candidate rootstock-regulated, fire blight susceptibility-associated transcripts, several pathways contained more genes than predicted relative to a KEGG analysis of all of the transcripts represented on the array (Table 2). Those pathways that had the highest confidence level for being over-represented include: fatty acid metabolism (ko00071), valine, leucine and isoleucine degradation (ko00280), photosynthesisantenna proteins (ko00196), flavonoid biosynthesis (ko00941), protein processing in the endoplasmic reticulum (ko04141), endocytosis (ko4144) and peroxisome (ko4146).


Page 6 of 17

Table 1 GO Enrichment Analysis Keyword Category

Functional Category

Expected

Observed

Biological Process

cell organization and biogenesis

29

29

Biological Process

developmental processes

22

21

Biological Process

DNA or RNA metabolism

8

2

Biological Process

electron transport or energy pathways

13

21

Biological Process

other biological processes

20

37*

Biological Process

other cellular processes

138

164

Biological Process

other metabolic processes

146

162

Biological Process Biological Process

protein metabolism response to abiotic or biotic stimulus

60 23

60 53*

Biological Process

response to stress

19

46*

Biological Process

signal transduction

15

7

Biological Process

transcription

23

4*

Biological Process

transport

31

31

Biological Process

unknown biological processes

124

33*

670

670

*Chi Square < 0.001

Genes differentially expressed during fire blight infection are disproportionately represented among the candidate rootstock-regulated, fire blight susceptibility-associated transcripts

Transcripts that increase or decrease in abundance during fire blight infection have been identified in apple [11-13]. Of the 690 candidate, rootstock-regulated, fire blight susceptibility-associated transcript sequences identified in the present study (Additional File 1, Tables S2 & S3), 79 (Table 3) had been determined to be differentially expressed during fire blight infection by Norelli et al., [11]. Based on BLAST searches of both sets of transcripts to the recently published apple genome [27], a number of the transcript sequences were found to represent different locations on the same predicted gene, resulting in a final set of 54 unique genes (Table 3). Based on the sizes of the candidate gene lists and the number of genes on the array and in the genome, approximately four genes are expected to be in common between the two data sets due to chance. Similarly, a total of 20 of our candidates corresponded to 10 genes identified by Baldo et al., [12] (Table 4), out of a total of 190 genes identified in that study. Half of these 20 candidates were also among those identified by Norelli et al [11]. We also compared our candidate list to a list of ~3,500 genes identified as being differentially expressed during flower infection by Sarowar et al., [13] and found that 117 of our candidates shared an Arabidopsis BLAST hit (Additional File 1, Table S4). For Baldo et al., and Sarowar et al., the number of genes expected to be in common between the two data sets due to chance are 2 and 60 respectively. Interestingly, of the 54 transcripts identified in common with Norelli et al., over half were down-regulated

post infection [11]. However, we found that these same genes had higher expression levels in the less susceptible trees. A few of these were initially up-regulated in the Norelli study at early time points, but were down-regulated at later time points. In Sarowar et al., again over half of the transcripts in common were identified as being down-regulated upon infection; they were expressed at higher levels in the least susceptible trees in the present study. Only 2 of 10 of the genes in common with Baldo et al. were down-regulated upon infection. A KEGG analysis of the genes in Table 3 is included in Table 2. Those pathways that had the highest confidence level for being overrepresented relative to the total transcriptome include protein processing in the endoplasmic reticulum (ko04141) and endocytosis (ko4144). Analysis of expression patterns of candidate fire blight susceptibility-associated genes in an apple rootstock breeding population

To further analyze which candidate rootstock-regulated, fire blight susceptibility-associated transcripts might prove to be the best indicators of resistance, we examined the expression levels of a subset of the candidate transcripts in a test population of 48 individual, non-grafted apple lines, grown in Geneva, NY, that were offspring from a single ‘Ottawa 3’ × ‘Robusta 5’ cross segregating for fire blight resistance. Expression was measured using a second-generation, 135,000 feature microarray (representing ~26,000 transcripts) developed from the original, larger microarray used for rootstock-regulated gene expression profiling [18]. The second-generation microarray was designed before the 690 fire blight-associated candidate transcripts had been identified; of the 690


Page 7 of 17

Table 2 KEGG functional analysis of candidate rootstock-regulated, fire blight susceptibility-associated transcripts

Pathway Fatty acid metabolism

Whole array

Suppl Table 2

Table 3 transcripts

Table 5 transcripts

KEGG no.

Total

Exp Obs

Exp

Obs

Exp

Obs

p-value > Suppl Table 2 Table 3 Table 5

71

10

0.1

4

n/a

n/a

n/a

n/a

0.001

n/a

n/a

Protein processing in endoplasmic reticulum

4141

60

0.8

8

0.3

3

0.3

3

0.001

0.001

0.001

Carbon fixation pathways in prokaryotes

720

10

0.1

3

n/a

n/a

n/a

n/a

0.001

n/a

n/a

Peroxisome

4146

30

0.4

5

0.2

1

n/a

n/a

0.001

0.05

n/a

Photosynthesis-antenna proteins

196

12

1.5

7

0.7

1

0.7

2

0.001

NS

0.001

Endocytosis

4144

30

0.4

3

0.2

2

0.2

1

0.001

0.001

0.05

Valine, leucine and isoleucine degradation

280

20

0.3

2

n/a

n/a

n/a

n/a

0.001

n/a

n/a

Carbon fixation in photosynthetic organisms

710

22

2.8

8

1.3

2

n/a

n/a

0.01

NS

n/a

Methane metabolism

680

19

2.4

7

1.1

2

n/a

n/a

0.01

NS

n/a

Spliceosome

3040

95

12.2

3

5.5

1

5.2

1

0.01

NS

NS

Arginine and proline metabolism

330

30

0.4

2

n/a

n/a

n/a

n/a

0.01

n/a

n/a

Flavonoid biosynthesis

941

13

1.7

5

0.7

2

0.01

Antigen processing and presentation

4612

6

0.8

3

0.3

2

0.3

2

0.05

0.01

0.01

Purine metabolism

230

75

9.6

2

n/a

n/a

n/a

n/a

0.05

n/a

n/a

Pentose and glucuronate interconversions

40

10

0.1

1

n/a

n/a

n/a

n/a

0.05

n/a

n/a

Indole alkaloid biosynthesis

901

1

0.1

1

n/a

n/a

n/a

n/a

0.05

n/a

n/a

Neuroactive ligand-receptor interaction

4080

1

0.1

1

n/a

n/a

n/a

n/a

0.05

n/a

n/a

Renin-angiotensin system

4614

1

0.1

1

n/a

n/a

n/a

n/a

0.05

n/a

n/a

Benzoate degradation

362

4

0.5

2

n/a

n/a

0.3

1

0.05

n/a

NS

Fructose and mannose metabolism

51

16

2.1

5

0.9

1

n/a

n/a

0.05

NS

n/a

NS

Ubiquitin mediated proteolysis

4120

47

6.0

1

n/a

n/a

3.2

1

0.05

n/a

NS

Cell cycle-yeast

4111

46

5.9

1

n/a

n/a

3.1

1

0.05

n/a

NS

Phagosome

4145

26

3.3

7

n/a

n/a

1.4

3

0.05

n/a

NS

NOD-like receptor signaling pathway

4621

3

0.4

1

0.2

1

0.2

1

NS

0.05

NS

NS = not significant, n/a = no transcripts identified matching this pathway.

candidate transcripts, 429 were represented on the second-generation microarray. Three different strains of E. amylovora (Ea2002a, Ea273a, and Ea4001a) were used to determine the susceptibility of the segregating population. Regression analysis of the data identified a set of 39 transcripts out of the 429 candidates that had some association with fire blight resistance in the breeding population (Table 5). The transcripts included in this set had un-adjusted pvalues of less than 0.05. For the transcripts identified as having higher levels of expression in less susceptible trees, 3 had an un-adjusted p-value below 0.05 for two strains, and 36 had an unadjusted p-value below 0.05 for one strain. For those transcripts that had higher expression levels in the more susceptible scion/rootstock combinations, only 3 transcripts had un-adjusted p-values below 0.05. The transcript list in Table 5 includes 3 transcripts previously shown to be differentially expressed upon E. amylovora infection [11] (Table 3). The functional annotation of these genes is included in Table 2. Those pathways, as

determined by the KEGG analysis, that had the highest confidence level for being disproportionately represented include: photosynthesis-antenna proteins (ko00196), protein processing in the endoplasmic reticulum (ko04141), and endocytosis (ko4144). Gene expression patterns correlating with fire blight susceptibility

A total of 13 genes with higher transcript levels in more resistant trees were identified as being significantly related to the level of fire blight caused by at least one of the three E. amylovora strains, with the resulting regression models accounting for between 40 and 60% of the variation in disease severity (Additional File 2, Table S5). Of these, increased transcription of one gene (APPLE0F000020273) was negatively related to levels of fire blight caused by all three strains. Increased levels of transcripts of APPLE0F000027501, APPLE00R00018643 and APPLE0F000019968 were related to lower levels of fire blight caused by at least two of the E. amylovora strains, while increased


Page 8 of 17

Table 3 Transcripts in common with those found to have differential expression in apple upon E. amylovora infection by Norelli et al., [11] Seq_ID

GenBank_Accn Malus Contig BLAST Hit

Description

APPLE0F000017044/

EG974017

MDC011793.214 plastocyanin-like domain-containing protein

EG974020

MDC019359.48

APPLE0F000022573

EH009495

MDC009164.88

APPLE0F000017734/

EH009489

MDC002049.218 leucine-rich repeat transmembrane protein kinase, putative

evalue SSH Response§ 9E-14

early upregulated

APPLE0FR00078469 APPLE0F000011437/ APPLE0F000051475/ APPLE0F000020924

fibrillarin 1 (FBR1) (FIB1) (SKIP7) identical to fibrillarin 1

1E-59

GI:9965653 from [A. thaliana]; C-terminus identical to SKP1

3E-53

interacting partner 7 GI:10716959 from [A. thaliana]

3E-28

putative cytochrome c oxidase subunit 5c [Helianthus annuus]

5E-14

upregulated 1 hpi

4E-11

upregulated 1 hpi

APPLE0F000060312

9E-06

APPLE0F000019504

EH009551

MDC011946.321 L-ascorbate peroxidase 1, cytosolic (APX1

APPLE0FR00043470

EH009570

MDC010527.333 Encodes a novel component essential for NDH-mediated nonphotochemical reduction of the plastoquinone pool in chlororespiratory electron transport.

APPLE0F000017575/

EH009572

MDC015841.261 oxygen-evolving enhancer protein 3, chloroplast, putative

EH009577

MDC006495.372 catalase 3 (SEN2)

EH009561/

MDC013772.152 putative strong similarity to plastidic fructose-bisphosphate)

APPLE0F000020381 APPLE0F000015792

(PSBQ2

APPLE0F000015821 APPLE0F000006776

early upregulated

7E-12

upregulated 1 hpi upregulated 1 hpi

5E-13

upregulated 1 hpi

7E-11 3E-12

upregulated 1 hpi

4E-16

EH009491

3E-25

upregulated 1 hpi

aldolase (EC 4.1.2.13) from Nicotiana paniculata (NPALDP1

APPLE00R00054041

EH009563

MDC008063.227 similar to putative 60S acidic ribosomal protein P0 GB:P50346

4E-32

upregulated 1 hpi

APPLE0F000022368

EH090785

MDC016063.220 Glycine-rich RNA-binding protein 7 {A. thaliana}

2E-11

upregulated 12 hpi

APPLE00R00018459

EH090787

MDC002341.180 40S ribosomal protein S27 (ARS27A

2E-17

upregulated 12 hpi

APPLE0F000016047/

EH090788

MDC013560.291 chlorophyll A-B binding protein 2, chloroplast/LHCII type I

5E-27

upregulated 12 hpi

1E-22

upregulated 2 hpi

APPLE0F000021251

CAB-2/CAB-140 (CAB2B)

APPLE00R00018530

EH090762

MDC009496.462 putative mRNA binding protein precursor (GI:26453355)

APPLE0F000021275

EH090763

MDC011225.39

APPLE0F000020273

EH090764

MDC007862.188 heat shock protein 81-4 (HSP81-4) contains Pfam profiles PF02518, 1E-22 PF00183: Hsp90 pro

dehydrin ERD10 (Low-temperature-induced protein LTI45) [A. thaliana] SWISS-PROT:P42759

upregulated 2 hpi upregulated 2 hpi


Page 9 of 17

Table 3 Transcripts in common with those found to have differential expression in apple upon E. amylovora infection by Norelli et al., [11] (Continued) APPLE0F000017683/

EG974803

MDC009596.270 sugar transporter, putative similar to ERD6 protein {A. thaliana}

EG974779

MDC021689.444 contains Pfam profile PF00407: Pathogenesis-related protein Bet v I family

1E-11

up-reg. 48 and 72 hpi

MDC007381.261 cytosolic (GAPC)/NAD-dependent glyceraldehyde-3-phosphate

1E-20


APPLE0F000019355 APPLE0F000015156

8E-29

1E-33

upregulated 48 and 72 hpi 72 hpi

APPLE0F000018467/ APPLE0F000019900/ APPLE0F000060942/ EG974812 APPLE0FR00073702/

dehydrogenase

APPLE0FR00045573/ APPLE0F000015746

EG974761

MDC017137.174 ribulose bisphosphate carboxylase small chain 3B/RuBisCO small subunit 3B (RBCS-3B) (ATS3B

8E-20


APPLE00R00062179

EH034496/

MDC006746.407 conserved hypothetical protein [Corynebacterium efficiens YS-

4E-07

early upreg., down-,

EG974019

314] mitochondrial 26S ribosomal RNA protein

reg. 2 hpi

APPLE00R00062069/ APPLE00R00062142/

early upreg., up-reg.

APPLE0FR00063223/

EH009493/

APPLE0FR00072001/ APPLE0FR00074986

EG974018

APPLE0F000019494/

EH034531/

APPLE0FR00070531

EH009567

APPLE0F000015761/

EH034470/

APPLE0F000016441/

EH034448/

APPLE0F000017178/

EH009539/

APPLE0F000016322

EH009499

MDC021568.167 3’utr of MDP0000053760 Unknown A. thaliana. protein

3E-12

1 hpi, down-reg. 2 hpi,

MDC008933.302 jasmonate-zim-domain protein 1

up-reg. 1 hpi, downreg. 2 hpi 1E-20

MDC012615.331 chlorophyll A-B binding protein 2, chloroplast/LHCII type I CAB-2/CAB-140 (CAB2B)

3E-70

up-reg. 1 hpi, down-

1E-20

reg. 2 hpi,

1E-70

EH034678/ EH034677/ EH034635/ APPLE00R00015736/

EH034631/

APPLE0F000025537

EH034620/

up-reg. 1 hpi, downMDC017026.232 ribulose bisphosphate carboxylase small chain 3B/RuBisCO

4E-26

small subunit 3B (RBCS-3B) (ATS3B)

reg. 1 hpi, down-reg. 12 hpi, down-reg. 24

EH034600/

hpi, downreg. 48

EH034581/

hpi,

EH034578/ EH009517/ EH009492 APPLE00R00061494

EH034644

MDC008988.418 ribosomal protein L2 family protein similar to ribosomal protein L2 4E-07 [Gossypium arboreum] GI:17644114; contains Pfam profile PF03947: Ribosomal Proteins L2, C-terminal domain

downregulated 1 hpi

APPLE00R00016015

EH034651

MDC013883.400 glycosyl hydrolase family 1 protein contains Pfam PF00232: domain; TIGRFAM TIGR01233: 6-phospho-beta-galactosidase; similar to amygdalin hydrolase isoform AH I precursor

downregulated 1 hpi

1E-22


Page 10 of 17

Table 3 Transcripts in common with those found to have differential expression in apple upon E. amylovora infection by Norelli et al., [11] (Continued) APPLE0F000019334

EH034681

MDC023674.27

APPLE0F000015900

EH034690

APPLE0FR00069028

EH034668/ EH034488/

expressed protein

4E-25

downregulated 1 hpi

MDC013694.297 68414.m02538 plastocyanin similar to plastocyanin GI:1865683 from [A. thaliana]

9E-11

downregulated 1 hpi

MDC019115.109 piggyBac transposable element derived 5 [Homo sapiens]

1E+00

down-reg. 1 hpi, down-reg. 2 hpi

MDC011711.503 elongation factor 1-alpha/EF-1-alpha

1E-81

down-reg. 1 hpi,

EH034454 APPLE0F000059582

EH034691/ EH034556

APPLE0F000021983

EH034459/

down-reg. 24 hpi MDC004849.508 sodium/calcium exchanger family protein/calcium-binding EF

EH034663 APPLE0F000017271

EH034459/

2E-18

downregulated 2 hpi

3E-48

downregulated 2 hpi

hand family protein contains Pfam profiles: PF01699 MDC004849.509 oxygen-evolving enhancer protein, chloroplast, putative/33 kDa

EH034663

subunit of oxygen evolving system of photosystem II,

APPLE0F000022030

EH034485

MDC015890.82

ADP-ribosylation factor, putative similar to DcARF1

3E-26

downregulated 2 hpi

APPLE0F000018523

EH034540

MDC015915.252 heat shock protein 70, putative/HSP70, putative strong similarity to 8E-49 heat shock protein GI:425194 [Spinacia oleracea]

downregulated 2 hpi

APPLE0F000020112

EH034525

MDC000302.731 ribulose bisphosphate carboxylase small chain 3B/RuBisCO small subunit 3B (RBCS-3B) (ATS3B)

7E-06

downregulated 2 hpi

EH034439

MDC021556.182 polyubiquitin (UBQ4) identical to GI:17677

1E-112 downregulated 2 hpi

APPLE0F000060823

EH034517

MDC012000.76

heat shock cognate 70 kDa protein 1 (HSC70-1) (HSP70-1)

APPLE0F000017293

EH034563/

MDC021812.77

peroxidase 42 (PER42) (P42) (PRXR1

APPLE0F000050108/ APPLE0F000061371/

APPLE0F000022561 downregulated 2 hpi 2E-57

EH034487

down-reg. 2 hpi, down-reg. 24 hpi

APPLE0F000017691

EH034538

MDC021024.27

phosphatidylinositol-4-phosphate 5-kinase family protein

APPLE0FR00045757

EH034536

MDC016554.129 possible 5’ utr for Thiamin diphosphate-binding fold (THDPbinding) superfamily protein

APPLE00R00002434/

EH034628

MDC008650.425 germin-like protein (GER3)

APPLE0F000018527

EH034604

MDC005688.181 oxygen-evolving enhancer protein, chloroplast, putative/33 kDa subunit of oxygen evolving system of photosystem II

APPLE00R00058028

EH034627

MDC010241.225 putative/L-iditol 2-dehydrogenase, putative similar to NADdependent sorbitol dehydrogenase from Malus x domestica

APPLE0F000049504

4E-51

down-reg. 24 hpi down-reg. 24 hpi

1E-62

down-reg. 48 hpi

9E-08 down-reg. 48 hpi 9E-26

down-reg. 48 hpi


Page 11 of 17

Table 3 Transcripts in common with those found to have differential expression in apple upon E. amylovora infection by Norelli et al., [11] (Continued) APPLE0FR00038628

EH009528

MDC000300.335 No Hits Found

APPLE0F000015847/

EH034662

MDC022487.75

upregulated 1 hpi

chlorophyll A-B binding protein/LHCII type I (LHB1B1)

5E-31

APPLE0F000016638

4E-40

APPLE0FR00073381

EH034693


APPLE0F000024130

EH034466

MDC004582.94

APPLE0F000059277

EG974791

MDC004462.498 60S ribosomal protein L19 (RPL19C) similar to L19 from several species

APPLE0FR00073380

EH034693


APPLE0FR00039729

EH009538


APPLE0FR00036633

EH009530

MDC001354.357 hypothetical protein [Burkholderia fungorum]

EG974808/ EG974767

MDC022200.129 catechol oxidase activity molecular function Catalysis of the reaction: 2 catechol + O2 metal ion binding molecular function Interacting selectively with any metal ion. predicted with glimmer

EH034619

MDC011650.665 expressed protein protein induced upon wounding-A. thaliana

protochlorophyllide reductase A, chloroplast/PCR A/NADPHprotochlorophyllide oxidoreductase A

5E-9 4E-41

.02

Up in sensitive APPLE0F000061746

APPLE0F000001556

up-reg. 48 and 72 hpi 4E-79

downregulated 48 hpi

§ SSH response corresponds to the change in expression after infection. Bold text corresponds to genes that are phosphorylated upon infection. Italicized text corresponds to genes that were also identified as differentially expressed upon infection by Baldo et al., [12]. Underlined text corresponds to genes that were also identified as differentially expressed upon infection by Sarowar et al., [13].

Table 4 Transcripts in common with those found to have differential expression in apple upon E. amylovora infection by Baldo et al., [12] SEQ_ID

GenBank_Accn

Malus Contig BLAST Hit

Description

evalue

cDNA-AFLP response§

APPLE0F000017734/

EX982051

MDC002049.218

leucine-rich repeat transmembrane protein kinase, putative”

4E-11

up-regulated 2

9E-06

hpi in M.26

235aa long hypothetical protein [Pyrococcus horikoshii]

3E-12

up-regulated 2

hypothetical protein [Deinococcus radiodurans]

5E-11

hpi in M.26

235aa long hypothetical protein [Pyrococcus horikoshii]

3E-12

down-regulated

hypothetical protein [Deinococcus radiodurans]

5E-11

2 hpi in M.26

235aa long hypothetical protein [Pyrococcus horikoshii] hypothetical protein [Deinococcus radiodurans]

3E-12 5E-11

up-regulated 2 & 48 hpi

APPLE0F000060312 APPLE00R00062069/ APPLE00R00062142/ APPLE0FR00063223/

EX982063

MDC021568.167

APPLE0FR00072001/ APPLE0FR00074986 APPLE00R00062069/ APPLE00R00062142/ APPLE0FR00063223/

EX982064

MDC021568.167

APPLE0FR00072001/ APPLE0FR00074986 APPLE00R00062069/ APPLE00R00062142/ APPLE0FR00063223/ APPLE0FR00063223/

EX982070

MDC021568.167

APPLE0FR00072001/

in M.26

APPLE0FR00074986 APPLE0F000062056

EX982066

MDC001431.265

APPLE0FR00045573/

EY437146

MDC005648.389

APPLE0FR00073702

hypothetical protein [Oenothera elata subsp. hookeri]

9E-18

up-regulated 2 hpi in G-41 up-regulated 48 hpi in G-41


Page 12 of 17

Table 4 Transcripts in common with those found to have differential expression in apple upon E. amylovora infection by Baldo et al., [12] (Continued) APPLE0F000020631/

EX982080

MDC005535.336 invertase/pectin methylesterase inhibitor family protein similar to pectinesterase from Arabidosis thaliana

EX982085

MDC003306.225

APPLE0F000016328/ APPLE0FR00076854/

plasma membrane intrinsic protein, putative very strong

up-regulated 48 hpi in G-41

7E-57

similarity to plasma membrane intrinsic protein (SIMIP)

APPLE0FR00076855

up-regulated 48

[Arabidopsis thaliana] GI:2306917”

APPLE00R00015876/

hpi in G-41 3E-19

APPLE0F000019554/ APPLE0F000021355/ APPLE0F000024114/

2E-19

4E-11 EX982096

MDC012593.380

chlorophyll A-B binding protein (LHCB2:4) nearly identical to Lhcb2 protein [Arabidopsis thaliana]

APPLE0FR00036952/

7E-16 5E-06

up-regulated 48 hpi in G-41

9E-18

down-regulated 48 hpi in G-41

GI:4741950

APPLE0FR00037537 APPLE0F000062056

EX982108

MDC001431.265

hypothetical protein [Oenothera elata subsp. hookeri] 3’ UTR of MDP499035

§ cDNA-AFLP response corresponds to the change in expression after infection Bold text corresponds to genes that are phosphorylated upon infection Italicized text corresponds to genes that were also identified as differentially expressed upon infection by Norelli et al., [11] Underlined text corresponds to genes that were also identified as differentially expressed upon infection by Sarowar et al., [13].

Table 5 Candidate transcripts whose expression levels correlated with fire blight resistance in a population of trees segregating for fire blight susceptibility (regression analysis). E. amylovora strain Seq_ID

E2002a Ea273 4001a

hit

Description

APPLE0F000021750

X

X

At3g54020

inositol phosphorylceramide synthase 2 -

evalue 3E-19

APPLE0F000017942

X

X

At5g17420

cellulose synthase, catalytic subunit (IRX3) identical to gi:5230423

5E-08

X

APPLE00R00017800

X

At5g02570

histone H2B, putative similar to histone H2B-2 Lycopersicon esculentum

2E-24

APPLE0F000016970

X

At3g01090

Snf1-related protein kinase (KIN10) (SKIN10)

2E-14

APPLE0F000020273*

X

At5g56000

heat shock protein 81-4 (HSP81-4)

APPLE0FR00077763

X

No Hits Found

APPLE0F000025563

X

At3g47520

APPLE0FR00039135

X

No Hits Found

APPLE0F000018055

X

At3g22550

APPLE0FR00077391

X

No Hits Found

APPLE0F000059277

X

At4g02230

APPLE0FR00069907

X

No Hits Found

APPLE0F000018558

X

At3g47470

APPLE0FR00078469

X

No Hits Found

APPLE0FR00072723

X

APPLE0FR00048629

1E-34 malate dehydrogenase [NAD], chloroplast (MDH) identical to chloroplast NAD-malate dehydrogenase [A. thaliana] GI:3256066 similar to senescence-associated protein SAG102

9E-30

4E-41 1E+00

60S ribosomal protein L19 2E-18 chlorophyll A-B binding protein 4, LHCI type III CAB-4 (CAB4)

1E-22

gb|AAD45359| AF161252_1

cycloidea-like protein [Linaria vulgaris]

2E-06

X

emb| CAG59326||

unnamed protein product [Candida glabrata CBS138] ref|XP_4463991| unnamed protein product [Candida glabrata]

2.E-02

P0671B1122 [Oryza sativa (japonica cultivar-group)]

2.E-06

APPLE0FR00038610

X

No Hits Found

APPLE00R00062246

X

dbj|BAB1270|

APPLE0F000021761

X

At1g72370

APPLE0FR00036516

X

gb|EAA23003|

APPLE0FR00076821

X

No Hits Found

APPLE0FR00076762

X

No Hits Found

APPLE0F000016441*

X

At1g29930

40S ribosomal protein SA (RPSaA) identical to laminin receptor-like protein 9.E-20 GB:U01955 [A thaliana] Ribosomal protein L31e, putative [Plasmodium yoelii yoelii]

2E-06

chlorophyll A-B binding protein 2, LHCII type I CAB-2/CAB-140

3.E-70


Page 13 of 17

Table 5 Candidate transcripts whose expression levels correlated with fire blight resistance in a population of trees segregating for fire blight susceptibility (regression analysis). (Continued) APPLE0F000019498

X

At2g42210

mitochondrial import inner membrane translocase subunit Tim17/Tim22/ Tim23 family protein

8E-07

APPLE0F000018523*

X

At3g12580

heat shock protein 70, putative/HSP70, putative

8E-49

APPLE00R00018643

X

At3g27690

chlorophyll A-B binding protein (LHCB2:4)

4E-22

APPLE0F000020073

X

At2g34690

expressed protein

3E-14

APPLE0F000021409

X

At3g05890

APPLE0FR00037149

X

No Hits Found

APPLE0FR00080491

X

gb|AAK07949| AF318573_29

unknown [Bovine herpesvirus 4]

1E+00

APPLE0F000025192

X

At3g56900

aladin-related/adracalin-related weak similarity to SP|Q9NRG9

3E-16

APPLE0F000060354

X

At5g02120

68418m00133 thylakoid membrane one helix protein (OHP)

1E+00

APPLE0F000001282

X

At4g38920

vacuolar ATP synthase 16 kDa proteolipid subunit 3/V-ATPase

1.E-34

APPLE0F000058071

X

At1g75780

tubulin beta-1 chain (TUB1) nearly identical to SP|P12411

2E-02

APPLE0F000020583

X

At2g34250

protein transport protein sec61, putative similar to PfSec61 [Plasmodium falciparum] GI:3057044

6E-34

APPLE0F000019736

X

At5g39740

60S ribosomal protein L5 (RPL5B) ribosomal protein L5, rice

2E-02

APPLE0FR00045816

X

No Hits Found

APPLE0FR00036516

X

gb|EAA23003|

Ribosomal protein L31e, putative [Plasmodium yoelii yoelii]

1E-16

APPLE0F000018069

X

At5g09810

actin 7 (ACT7)/actin 2 identical to SP|P53492 Actin 7 (Actin-2) {Arabidopsis 4E-72 thaliana}

X

At1g04540

C2 domain-containing protein low similarity to cold-regulated gene SRC2 3E-11 [Glycine max]

X

ref|XP_032996|

similar to KIAA0819 protein [Homo sapiens]

3E-01

sp|P43309| PPO_MALDO

Polyphenol oxidase, chloroplast precursor (PPO) (Catechol oxidase)

0E+00

APPLE0F000026657# APPLE0FR00032503# APPLE0F000061746#

X

hydrophobic protein (RCI2B)/low temperature and salt responsive protein 5E-10 (LTI6B)

X = an un-adjusted p value of less than 0.05 for the analysis * Transcripts also found in Table 3 # Transcripts less abundant in plants with low susceptibility to fire blight Underlined text corresponds to genes that were also identified as differentially expressed upon infection by Sarowar et al., [13].

transcript levels of APPLE0F000019334, APPLE0F0 00018558, APPLE0F000020583 and APPLE0F0000 23953 were noted in tissues with higher levels of disease for at least two of the strains (Additional File 2, Table S5). The models relating the expression of genes with higher levels of expression in more susceptible trees to levels of fire blight were much weaker, only accounting for between 29 and 32% of disease severity (0.031 ≤ P ≤ 0.009; Additional File 2, Table S6). In all, lower expression levels of eight of the genes were related to levels of fire blight for at least one of the E. amylovora strains, with APPLE0FR00081295, APPLE0FR00067567 and APPLE0F000016771 being related with higher levels of fire blight caused by Ea 273 (Additional File 2, Table S6). Only the lower transcript levels of APPLE0F000026657 were related to lower levels of fire blight for all three E. amylovora strains, while lower transcript levels of APPLE0FR00066754 and APPLE0FR00063520 were associated with lower levels of disease caused by Ea273 and Ea4001a, respectively (Additional File 2, Table S6).

Discussion In this study, we found that rootstock genotype influenced ‘Gala’ scion fire blight susceptibility in grafted apple trees. This indicates that at least some level of resistance possessed by the rootstock can be conferred upon the scion variety that is grafted to it. These phenotypic differences in scion fire blight susceptibility were associated with reproducible patterns of gene expression in uninfected trees. Most of the transcripts identified in this study had higher levels of expression in the least susceptible trees. The expression levels of some of these genes may play a role in determining the susceptibility status of apple trees to E. amylovora prior to infection. Some of the identified genes may also play a role in fire blight disease resistance after infection has begun. It is also possible that some of the genes identified in the study affect the suitability of the host environment for the bacterium, rather than being involved in defense directly. Tree breeding is a slow and costly process, particularly due to long juvenile periods. The screening of seedlings for the expression of a suite of genes correlated with a


given trait could provide a valuable short-cut to reduce breeding time. The suite of genes identified in the present study might be useful as predictors of the fire blight resistance status of apple trees and seedlings. Seedlings could be selected based on gene expression patterns associated with favorable traits. This approach is not novel; the use of expression-based markers has proven to be effective in the screening of human breast cancers in order to predict the aggressiveness of the tumor [28]. Some of the genes identified in this study might also be suitable targets for direct manipulation for improvement of apple tree fire blight resistance and for the development of sequence-based molecular breeding markers. Pathways and processes

Three transcripts on the array corresponding to genes in the phenylpropanoid pathway were on our preliminary list of rootstock-regulated candidates, including two for chalcone synthase (APPLE0F000017774, APPLE0F0000 178640) and a chalcone isomerase (APPLE0F000056938) (Additional File 1, Table S2). However, these genes do not appear on any of our subsequent lists. Nevertheless, the expression pattern of the genes in the phenylpropanoid pathway as a whole (Figure 2) is consistent with the proposed role of this pathway in the response to E. amylovora infection [14,15]. This suggests that the expression of the phenylpropanoid pathway as a whole might be a good predictor of fire blight resistance. Sorbitol dehydrogenase (SDH) (APPLE0F000058028, APPLE0F000007408, APPLE0F000017030), was found to be expressed at higher levels in the trees that were least susceptible to fire blight (Table 3 and Additional File 1, Table S4). Sorbitol is a major form of translocated sugar in apples [29]. SDH converts sorbitol to fructose in sink tissues [30]. For E. amylovora, sorbitol is an important factor in determining host specificity [31]. It may be that higher SDH levels reduce the availability of sorbitol to E. amylovora. However it has also been shown that high sorbitol levels can inhibit the development of disease symptoms [32]. Our analysis of the recently released apple genome suggests that there may be up to 28 genes encoding SDH enzymes, as opposed to a single copy of SDH in Arabidopsis (AT5G51970). This complexity points to the importance of SDH to apple physiology. Not surprisingly, there was a GO annotation enrichment among the genes having higher expression in the least susceptible trees (Table 1), with genes predicted to be involved in responses to stress and biotic and abiotic stimuli being disproportionately represented relative to the known apple transcriptome as a whole. Upon further analysis of these transcripts (Table 2), more transcripts than expected, relative to the proportion among all of the transcripts represented on the array, were identified in the secretory pathway, including

Page 14 of 17

several heat shock proteins (APPLE0F000018523, APPLE0F000020273, APPLE0F000060823), suggesting that protein processing in the endoplasmic reticulum may be more active in the trees least susceptible to fire blight. Heat shock proteins are important for protein processing in the endoplasmic reticulum and have also been shown to play critical roles in signal transduction in defense responses in tobacco [33]. Additionally, higher levels of the transcripts encoding a predicted calnexin (APPLE0F000027501) and a Sec61 homolog (APPLE0F000020583) were associated with reduced fire blight susceptibility by stepwise multiple regression analysis. Both of these proteins have functional annotations indicating involvement in protein processing in the endoplasmic reticulum. Consistent with these findings, it has been shown that the entire secretory pathway is up-regulated during systemic acquired resistance (SAR) [34] as well as during the N-mediated viral defense response in tobacco [35]. Similarly, from the same data set, a greater than expected number of genes involved in both the endocytosis and peroxisomal pathways were identified, suggesting that these processes may be more active in the least susceptible trees. Intriguingly, endocytosis processes are involved in plant recognition of pathogen-associated molecular patterns (PAMPs) [36]. For example, the bacterial flagellin protein FLG22 is recognized in plants by the FLS2 receptor kinase [37], which leads to endocytosis of the FLS2 receptor and its subsequent degradation by the proteosome [38]. Higher levels of a transcript encoding a predicted fatty acyl CoA ligase (APPLE0F000019968) were associated with reduced fire blight susceptibility by the stepwise multiple regression analysis. The protein encoded by this gene has a functional annotation indicating involvement in endocytosis and fatty acid metabolism. We also identified a transcript encoding a putative phosphatidylinositol-4-phosphate 5-kinase (PIP5K) (APPLE0F000 017691) that had higher expression levels in the least susceptible trees. The product of PIP5K, phosphoinositol 4,5-bisphosphate (PI(4,5)P2), is a key regulator of clathrinmediated endocytosis [39]. Additional candidate genes of particular interest

A major objective of this study was to identify candidate genes in apple that are potentially involved in determining fire blight resistance prior to an infection event. Interestingly, quite a few of the candidate genes identified in our study had previous links to disease resistance processes, including differential expression upon E. amylovora infection in apple [11-13]. This includes several heat shock proteins, a leucine-rich repeat transmembrane kinase, and sorbitol dehydrogenase. Strikingly, over half of the transcripts that we identified as being expressed at higher levels in less susceptible trees are down-regulated during


E. amylovora infection [11-13]. It is possible that the expression of these genes is down-regulated by the pathogen to promote disease. Additionally, eight of the genes in both Tables 3 have also been shown to be phosphorylated upon infection [40], which offers another level of regulation in addition to changes in transcript abundance. Only one gene was found in common between our data set and those of Norelli et al. [11], Baldo et al. [12], and Sarowar et al [13]. This gene had higher steady-state expression levels in resistant trees and was up-regulated in all three pathogen induction studies. The Arabidopsis homolog of APPLE0F000017734/APPLE0F000060312 (AT2G31880 or SOBIR1) (Tables 3 and 4, Additional File 1, Table S4) encodes a putative leucine rich repeat transmembrane protein that is expressed in response to Pseudomonas syringae infection in Arabidopsis. Overexpression of SOBIR1 in Arabidopsis caused a constitutive upregulation of PR-1 and PR-2, and the plants showed enhanced resistance to P. syringae DC3000, suggesting that elevated levels of SOBIR1 lead to a constitutive activation of disease-resistance responses [41]. SOBIR1 overexpression also resulted in the activation of cell death. It has been proposed that SOBIR1 may play a role in the regulation of the golgi apparatus, particularly during periods of cellular stress [42]. We also identified a transcript encoding a putative cell death regulator, inositolphosphorylceramide synthase (APPLE0F000021750, Additional File 1, Table S4), that had higher expression levels in the least susceptible trees. Inositol can be modified by inositolphosphorylceramide synthase to produce inositolphosphorylceramide. Inositolphosphorylceramide has been shown to be involved in the regulation of programmed cell death during the plant defense response [43]. Jasmonic acid has been shown to play an important role in the response to pathogens, in a pathway parallel to that of salicylic acid [44,45]. In particular, ethylene and jasmonate have been shown to play an important role in defense against necrotrophic pathogens like E. amylovora [46]. We also identified homologs of the JAZ1 gene in Arabidopsis (APPLE0F000019494/APPLE0F000070531, Table 3, Additional File 1, Table S4) that had greater expression in the least sensitive trees. The JAZ1 protein of Arabidopsis is part of the COI1/JAZ jasmonate receptor complex [47].

Conclusions The influence of rootstocks on the fire blight susceptibility and gene expression of the scion has allowed us to identify genes potentially associated with this phenotypic trait. The identification of these genes will contribute to the understanding of host-pathogen interactions as well as provide plant breeders with valuable new markers for improved disease resistance breeding. This study illustrates the utility of our rootstock-regulated gene

Page 15 of 17

expression data sets for candidate trait-associated gene data mining.

Additional material Additional file 1: Tables S1-S4. Table S1. Expression patterns of putative phenylpropanoid genes from Malus x domestica. Table S2. Candidate fire blight resistance transcripts with higher expression in ‘Gala’ scion/rootstock combinations with lower susceptibility to E. amylovora. Table S3. Candidate fire blight resistance transcripts with higher expression in ‘Gala’ scion/rootstock combinations with higher susceptibility to E. amylovora. Table S4. Transcripts in common with those known to have differential expression in apple flowers upon E. amylovora infection [13] (Sarowar et al. 2011). Additional file 2: Tables S5-S6. Table S5. Stepwise multiple regression analysis parameter estimates and associated statistics for transcripts with higher expression in less susceptible trees. Table S6. Stepwise multiple regression analysis parameter estimates and associated statistics for transcripts with higher expression in more susceptible trees.

Acknowledgements This project was funded by NSF Plant Genome Research Program Grant No. 04-20394 to TWM, SNM, JWT and RMC. We thank Candace L. Price for performing sample labeling and hybridization of our second-generation DNA microarrays. Author details Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA. 2The Pennsylvania State University Fruit Research and Extension Center, Biglerville, PA 17307, USA. 3USDA/ARS, Plant Genetics Research Unit, Geneva, NY 14456, USA. 4Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland. 5Department of Statistics, The Pennsylvania State University, University Park, PA 16802, USA. 6Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA. 7Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA. 1

Authors’ contributions TWM conceived the study and participated in its design and coordination. PJJ performed plant material preparation and mRNA extraction for microarray hybridizations. PJJ and NA carried out the computational analysis of the microarray data. NH and JWT conducted the fire blight susceptibility field study in Biglerville, PA. GF provided the tree samples and fire blight data from Geneva, NY. IM developed the contig sequences used for the microarray. CP and SNM assisted in the experimental design. CP performed the second-generation microarray hybridizations. RMC contributed to the experimental design, selection of rootstocks and general culture of trees in the field. HKN performed the multiple regression analysis of the gene expression data. PJJ and TWM analyzed the computational results and drafted the manuscript. All authors commented on the manuscript and approved the final version. Competing interests The authors declare that they have no competing interests. Received: 8 July 2011 Accepted: 9 January 2012 Published: 9 January 2012 References 1. Burill TJ: Anthrax of fruit trees; or the so-called fire blight of pear, and twig blight of apple trees. Am Ass Adv Sci Proc 1880, 29:583-597. 2. McManus PS, Stockwell VO, Sundin GW, Jones AL: Antibiotic use in plant agriculture. Annual Review of Phytopathology 2002, 40:443-465. 3. Agrios GN: Plant Pathology. 4 edition. California: Academic Press; 1997.


4.

5. 6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17. 18.

19.

20. 21.

22. 23. 24.

25.

Autio WR, Southwick FW: The effects of rootstock and root-interstem combination on the growth, productivity, and anchorage of a spur and standard strain of delicious apple tree. Fruit Varieties Journal 1986, 40(4):128-133. Wilcox WF: Fire blight fact sheet. 1994 [http://www.nysipm.cornell.edu/ factsheets/treefruit/diseases/fb/fb.pdf]. Carlson RF: Rootstocks in relation to apple cultivars. In North American Apples: Varieties, Rootstocks, Outlook. Edited by: Carlson RF, Degman ES, French AP, Larsen RP, Maas V, Mowry JB, Rollins HA, Upshall WH, Wilcox E. East Lansing, MI: Michigan State University Press; 1970:153-180. Cline JA, Hunter DM, Bonn WG, Bijl M: Resistance of the Vineland series of apple rootstocks to fire blight caused by Erwinia amylovora. Journal American Pomological Society 2001, 55(4):218-221. Maxson-Stein K, He SY, Hammerschmidt R, Jones AL: Effect of treating apple trees with acibenzolar-S-methyl on fire blight and expression of pathogenesis-related protein genes. Plant Disease 2002, 86(7):785-790. Bonasera JM, Kim JF, Beer SV: PR genes of apple: identification and expression in response to elicitors and inoculation with Erwinia amylovora. BMC Plant Biology 2006, 6:23. Malnoy M, Jin Q, Borejsza-Wysocka EE, He SY, Aldwinckle HS: Overexpression of the apple MpNPR1 gene confers increased disease resistance in Malus x domestica. Molecular Plant-Microbe Interactions 2007, 20(12):1568-1580. Norelli JL, Farrell RE, Bassett CL, Baldo AM, Lalli DA, Aldwinckle HS, Wisniewski ME: Rapid transcriptional response of apple to fire blight disease revealed by cDNA suppression subtractive hybridization analysis. Tree Genetics & Genomes 2009, 5(1):27-40. Baldo A, Norelli JL, Farrell RE Jr, Bassett CL, Aldwinckle HS, Malnoy M: Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus x domestica) with Erwinia amylovora. BMC Plant Biology 2010, 10. Sarowar S, Zhao Y, Soria-Guerra RE, Ali S, Zheng D, Wang D, Korban SS: Expression profiles of differentially regulated genes during the early stages of apple flower infection with Erwinia amylovora. J Exp Bot 2011. Venisse JS, Malnoy M, Faize M, Paulin JP, Brisset MN: Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora. Molecular Plant-Microbe Interactions 2002, 15(12):1204-1212. Milcevicova R, Gosch C, Halbwirth H, Stich K, Hanke MV, Peil A, Flachowsky H, Rozhon W, Jonak C, Oufir M, et al: Erwinia amylovorainduced defense mechanisms of two apple species that differ in susceptibility to fire blight. Plant Science 2010, 179(1-2):60-67. Frick S, Kutchan TM: Molecular cloning and functional expression of Omethyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis. Plant Journal 1999, 17(4):329-339. Whetten R, Sederoff R: Lignin biosynthesis. Plant Cell 1995, 7(7):1001-1013. Jensen PJ, Makalowska I, Altman N, Fazio G, Praul C, Maximova SN, Crassweller RM, Travis JW, McNellis TW: Rootstock-regulated gene expression patterns in apple tree scions. Tree Genetics & Genomes 2010, 6(1):57-72. Fazio G, Wan Y, Russo NL, Aldwinckle HS: Investigation on the inheritance of strain specific resistance to Erwinia amylovora in an apple rootstock segregating population. In Proceedings of the Sixth International Workshop on Fire Blight: 12-17 August 2007. Edited by: Johnson KB, Stockwell VO. Portland, OR: ISHS Acta Horticulturae; 2008:331-336. Gene Expression Omnibus (GEO) dataset website:[http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GPL3715]. Irizarry RA, Hoobs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostat 2003, 4:249-264. Bioconductor:[http://www.bioconductor.org/]. Afifi AA, Clark V, May S: Computer-Aided Multivariate Analysis Fourth Edition Boca Raton, FL: Chapman & Hall/CRC; 2004. Lee SA, Ngugi HK, Halbrendt NO, O’Keefe G, Lehman B, Travis JW, Sinn JP, McNellis TW: Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings. Phytopathology 2010, 100(6):539-550. AraCyc Pathways:[http://www.arabidopsis.org/biocyc/index.jsp].

Page 16 of 17

26. KEGG Automatic Annotation Server:[http://www.genome.jp/tools/kaas/]. 27. Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar SK, Troggio M, Pruss D, et al: The genome of the domesticated apple (Malus x domestica Borkh.). Nature Genetics 2010, 42(10):833-839. 28. van’t Veer LJ, Dai HY, van de Vijver MJ, He YDD, Hart AAM, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, et al: Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002, 415(6871):530-536. 29. Bieleski RL: Accumulation and translocation of sorbitol in apple phloem. Australian Journal of Biological Sciences 1969, 22(3):611-620. 30. Negm FB, Loescher WH: Detection and characterization of sorbitol dehydrogenase for apple callus-tissue. Plant Physiology 1979, 64(1):69-73. 31. Aldridge P, Metzger M, Geider K: Genetics of sorbitol metabolism in Erwinia amylovora and its influence on bacterial virulence. Molecular & General Genetics 1997, 256(6):611-619. 32. Suleman P, Steiner PW: Relationship between sorbitol and solute potential in apple shoot relative to fire blight symptom development after infection by Erwinia amylovora. Phytopathology 1994, 84(10):1244-1250. 33. Kanzaki H, Saitoh H, Ito A, Fujisawa S, Kamoun S, Katou S, Yoshioka H, Terauchi R: Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Molecular Plant Pathology 2003, 4(5):383-391. 34. Wang D, Weaver ND, Kesarwani M, Dong XN: Induction of protein secretory pathway is required for systemic acquired resistance. Science 2005, 308(5724):1036-1040. 35. Caplan JL, Zhu XH, Mamillapalli P, Marathe R, Anandalakshmi R, DineshKumar SP: Induced ER chaperones regulate a receptor-like kinase to mediate antiviral innate immune response, in plants. Cell Host & Microbe 2009, 6(5):457-469. 36. Gordon S: Pattern recognition receptors: Doubling up for the innate immune response. Cell 2002, 111(7):927-930. 37. Gomez-Gomez L, Boller T: FLS2: An LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Molecular Cell 2000, 5(6):1003-1011. 38. Robatzek S, Chinchilla D, Boller T: Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes & Development 2006, 20(5):537-542. 39. Simonsen A, Wurmser AE, Emr SD, Stenmark H: The role of phosphoinositides in membrane transport. Current Opinion in Cell Biology 2001, 13(4):485-492. 40. Jones AME, Bennett MH, Mansfield JW, Grant M: Analysis of the defence phosphoproteome of Arabidopsis thaliana using differential mass tagging. Proteomics 2006, 6(14):4155-4165. 41. Gao MH, Wang X, Wang DM, Xu F, Ding XJ, Zhang ZB, Bi DL, Cheng YT, Chen S, Li X, et al: Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis. Cell Host & Microbe 2009, 6(1):34-44. 42. Leslie ME, Lewis MW, Youn JY, Daniels MJ, Liljegren SJ: The EVERSHED receptor-like kinase modulates floral organ shedding in Arabidopsis. Development 2010, 137(3):467-476. 43. Wang WM, Yang XH, Tangchaiburana S, Ndeh R, Markham JE, Tsegaye Y, Dunn TM, Wang GL, Bellizzi M, Parsons JF, et al: An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis. Plant Cell 2008, 20(11):3163-3179. 44. Thomma B, Eggermont K, Penninckx I, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF: Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(25):15107-15111. 45. Vijayan P, Shockey J, Levesque CA, Cook RJ, Browse J: A role for jasmonate in pathogen defense of Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(12):7209-7214.


Page 17 of 17

46. Glazebrook J: Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology 2005, 43:205-227. 47. Sheard LB, Tan X, Mao HB, Withers J, Ben-Nissan G, Hinds TR, Kobayashi Y, Hsu FF, Sharon M, Browse J, et al: Jasmonate perception by inositolphosphate-potentiated COI1-JAZ co-receptor. Nature 2010, 468(7322):400-405. doi:10.1186/1471-2164-13-9 Cite this article as: Jensen et al.: Rootstock-regulated gene expression patterns associated with fire blight resistance in apple. BMC Genomics 2012 13:9.

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit